The overall goal of the laboratory is to bring novel technologies and approaches to life science and biological research in order to close the gap that exists between the bench and the bedside. Specifically, research in the laboratory is focused on the investigation the role of a novel, oxygen-dependent negative regulator of the hearts response to ischemia. The identification of this novel oxygen-dependent regulator of HIF may provide additional therapeutic avenues when treating ischemic heart disease. Additional projects in the laboratory involve the development of a custom biosensor testing platform for the detection of blood-based biomarkers, as well as design and testing of a novel gene therapy delivery platform based on custom ultrasound equipment.
Mark Khemmani (Technician)
Brandyn Dunn (Medical Student)
Rajander Nirwan (Medical Student)
Brandon Kobayashi (Undergraduate; Microbiology)
Cynthia Anderson (Ph.D. student, Thesis advisor: Dr. Ralph V. Shohet)
1. HIF-1 Overexpression
Hypoxia-inducible factor 1 (HIF-1) regulates hundreds of genes that mediate the cellular response to hypoxia. The downstream physiologic effects of HIF-1 are wide-ranging and include effects on iron metabolism, angiogenesis, glycolytic metabolism, and cell survival. Clinical pathologies involving HIF-1 are equally broad [1-3]. These include abnormal embryonic vasculogenesis, ischemic disease of the heart and cerebrovasculature, Von Hippel Lindau syndrome, and cancer angiogenesis. Given the important role of oxygen signaling pathways in the pathogenesis of these diseases, there is substantial clinical interest in factors that regulate HIF.
We have previously investigated the effects of HIF-1α in the heart by expressing an oxygen-stable form specifically in cardiac myocytes. In our evaluations, we have identified a novel protein, which negatively regulates the activity of HIF-1. While the role of this novel protein in normal physiologic response to hypoxia remains to be investigated, the phenotypic changes seen with this novel interaction with oxygen-stable HIF may open the doors for other potential targets of anti-ischemic and anti-angiogenesis treatments.
In collaboration with the Shohet laboratory, we are working out the mechanism of neoangiogenesis seen with stable cardiac HIF-1α expression. Gross inspection of the hearts after overexpression demonstrates enlarged hearts with large epicardial vessels and prominent side branches. Perfusion curve analyses demonstrate an increase in the myocardial red cell volume after HIF-1α expression. Corrosion casts of cardiac vessels made with a new low-viscosity resin that can fill the vasculature down to the level of the capillaries, reveal “lakes” of capillaries forming off of larger vessels after HIF expression (Fig 2). Pro-angiogenic factor evaluations support a pattern of progressive functional neoangiogenesis. Taken together this demonstrates the structural characteristics of HIF-directed angiogenesis and supports the utility of manipulation of HIF signaling to enhance perfusion and treat ischemia.
Figure 2. Corrosion casts of coronary vasculature. (A and C) one day after doxycycline removal, (B) tTA+/HIF-1α-PPN+ mice maintained on doxycycline, and (D) seven days off doxycycline. All SEM images focus on coronary vessels.
(click on image to enlarge)
3. Biosensor and point-of-care diagnostic development
The Center for Cardiovascular Research has been working on the proof-of-
concept and feasibility testing for a patented and patent-pending ultrasensitive electrical biosensor (Fig 3) in collaboration with its inventor, Professor James Holm-Kennedy, for the last 4 years.
Figure 3. Layout of biosensor
We are developing the biosensor into a blood-based, cardiac troponin I-specific diagnostic tool for discriminating between cardiac angina and acute coronary syndromes. The ability to measure the cTI levels to a more sensitive level is not only crucial for determining the difference between angina and more severe ACS, but will be a useful clinical tool for many cardiac events, and allow for a more focused direction of appropriate clinical intervention, improving patient outcomes. Low cost supporting instrumentation can be conventional off-the shelf electronic instruments and we are currently developing a prototype point-of-care device (Fig 4).
Additionally, our biosensor provides a leveraged technology return since our same sensing approach can be applied to other targets (e.g., viruses, proteins, nucleic acids, etc.).
4. Ultrasound projects
An on-going project in the laboratory is the use of ultrasound as a platform gene transfer platform technology, termed Ultrasound Targeted Microbubble Destruction. Ultrasound targeted microbubble destruction (UTMD) is a technique for transformation in which bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents  (Fig 5A).
In vivo these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In vitro, the microbubbles can simply be added to the media containing the cells of interest. The DNA delivery to the target cell occurs by acoustic cavitation at a resonant frequency of the microbubbles (Fig 5B). It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in the cells. As a result of this sonoporation effect, the transfection efficiency into and across the cells is enhanced. We have recently finished the design and fabrication of a custom ultrasound testing device allowing us the ability to modulate all ultrasonic parameters needed to optimize delivery of genes to any target organ/tissue including; brain, heart, placenta, liver and testes. An additional project involves using ultrasound to deliver genes to microalgae with an interest in expressing commercial important products such as vaccines and active enzymes. Uses include: biofuels, pharmaceuticals, aquaculture feed, etc.
Dr. Chad Walton